Method of modifying oligosaccharide structure of tissue plasminogen activator

ABSTRACT

A method is disclosed for modifying a glycoprotein&#39;s oligosaccharide structure by substituting or deleting an amino acid residue at a position which is remote from the glycosylation site.

BACKGROUND OF THE INVENTION

This invention relates to a method of modifying a glycoprotein'soligosaccharide structure.

Many proteins of biological and pharmaceutical interest haveoligosaccharides attached to their polypeptide backbone. Althoughproteins produced by E. coli and other bacteria are non-glycosylated,proteins secreted by yeasts and mammalian cells are normallyglycosylated. The sugar chains of these glycoproteins can be attached byan N-glycosidic bond to the amide group of asparagine residues(Asn-linked oligosaccharides) or by an O-glycosidic bond to the hydroxylgroup of serine or threonine residues (Ser- or Thr-linkedoligosaccharides).

In the Asn-linked oligosaccharides, the types of structures found onnative proteins can be generally classified as high mannose, hybrid andcomplex type sugar chains. However, considerable variation in thesebasic structures is common. See, for example, the 16 oligosaccharidestructures on a tissue plasminogen activator derived from normal humancolon fibroblast cells as described in U.S. Pat. No. 4,751,084. Furtherbackground information on the assembly of Asn-linked oligosaccharidescan be had by reference to Kornfeld and Kornfeld, Ann. Rev. Biochem. 54,631-664 (1985).

The carbohydrate structure of a glycoprotein can have a significanteffect upon its biological activity. That is, the oligosaccharides canaffect the protein's antigenicity, stability, solubility and tertiarystructure. The carbohydrate side-chains also can influence the protein'shalf-life and target it to receptors on the appropriate cells. Thecarbohydrate residues can affect both inter- and intracellularrecognition. The sugar groups thus can control the relativeeffectiveness of a therapeutic protein when administered to a patient.These and other such functions of the carbohydrate moiety ofglycoproteins are discussed, for example, by Delente, Trends in Biotech.3(9), 218 (1985); van Brunt, Bio/Technology 4, 835-839 (1986); andTaunton-Rigby, Biotech USA 1988, Proc. Conf. San Francisco, Nov. 14-16,1988, pp. 168-176.

It is also apparent that differences in the glycosylation pattern (i.e.,particular structure at a specific site) on similar proteins or proteinswith identical amino acid sequences can have profound effects onantigenicity, metabolism and other physiological properties. See, forexample, the association of rheumatoid arthritis and osteoarthritis withchanges in the glycosylation pattern of total serum by Parekh et al.,Nature 316, 452-457 (1985) and in U.S. Pat. No. 4,659,659.

Another example of a glycoprotein in which significant biologicalactivity resides in the oligosaccharide moieties is that of humanchorionic gonadotropan (hCG). Thus, it is known that hCG withoutcarbohydrate is a competitive inhibitor of native hCG; thatoligosaccharides isolated from hCG inhibit action of native hCG; andthat tumor-produced hCG having the same amino acid sequence as nativehCG but different sugars has almost no biological activity. See Calvo etal., Biochemistry 24, 1953-1959 (1985); Chen et al., J. Biol. Chem. 257,14446-14452 (1982).

Yet another group of proteins in which the presence and/or structure ofthe oligosaccharides can have important biological effects are theplasminogen activators (PA), namely urokinase (u-PA) and tissueplasminogen activator (t-PA). The functional properties ofcarbohydrate-depleted t-PA are discussed ty Little et al., Biochemistry23, 6191-6195 (1984), and by Opdenakker et al., "EMBO Workshop onPlasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985. The latterscientists report that enzymatic cleavage of carbohydrate side-chainsfrom melanoma (Bowes) derived t-PA by treatment with α-mannosidasecauses an increase in the biologic activity of the modified t-PA. TheBowes melanoma t-PA is a glycoprotein which has a molecular weight ofabout 68,000-70,000 daltons and a 527 amino acid structure with serineat the NH₂ -terminus. The melanoma t-PA can exist as two chains, anA-chain and a B-chain. It also separates into two variants (or isoforms)in the A-chain, known as types I and II, which differ by about M_(r)2000-3000. See Ranby et al., FEBS Lett. 146 (2), 289-292 (1982), andWallen et al., Eur. J. Biochem. 132, 681-686 (1983). Type I isglycosylated at Asn-117, Asn-184 and Asn-448, whereas Type II isglycosylated only at Asn-117 and Asn-448 according to Pohl et al.,Biochemistry 23, 3701-3703 (1984). A high mannose structure has beenassigned to Asn-117, whereas two complex carbohydrate structures areassigned to Asn-184 and Asn-448 by Pohl et al., "EMBO Workshop onPlasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985, and Eur. J.Biochem. 170, 69-75 (1987).

It is known that the normal t-PA molecule has five functional domains orregions: A fibronectin-like finger domain (F); an epidermal growthfactor region (GF); two kringle regions (K1 and K2); and a serineprotease region (SP). The full t-PA molecule thus can be represented asF+GF+K1+K2+SP. In the 527 amino acid sequence of the normal t-PAmolecule described by Pennica et al., Nature 301, 214-221 (1983), thefinger region comprises residues 1-43; the growth factor regioncomprises residues 44-91; kringle refers to a characteristic tripledisulfide structure of which t-PA has two such regions, K1 - residues92-173, and K2 - residues 180-261; and the serine protease comprisesresidues 262-527. The SP catalytic site is formed from the His-322,Asp-371 and Ser-478 residues. Various deletions of one or more of theseregions together with elimination of one or more of the glycosylationsites such as by site-directed mutagenesis have been describedheretofore.

In European Patent Application 178,105, published Apr. 16, 1986, amodified t-PA is described in which one or more of the glycosylationsites have been eliminated by site-directed mutagenesis of Asn to Gln atthe glycosylation sites in the kringle and serine protease regions. Theamino acid residues Asn-120, -187 and -451 in the described uterine t-PAare equivalent to residues Asn-117, -184 and -448, respectively, in theBowes melanoma t-PA.

A variety of site-mutagens are also described in European PatentApplication 227,462, published July 1, 1987, including mutagenesis atthe above glycosylation sites and at the cleavage sites in the region272-280, especially in the sequence Phe(274)-Arg(275)-Ile(276)-Lys(277).

According to European Patent Application 238,304, published Sept. 23,1987, melanoma t-PA devoid of carbohydrate structure at amino acidresidue 117 but unmodified from native t-PA in functional carbohydratestructure at amino acid residues 184 and/or 448 retains substantiallyfull biological activity compared to native t-PA but has increased invivo half-life. See also Hotchkiss et al., Thromb. Haemostasis 60,255-261 (1988).

In U.S. Pat. No. 4,751,084, a glycosylated t-PA obtained from culturednormal human colon fibroblast cells was found to have a unique,heterogeneous glycosylation pattern that differs significantly from thet-PA of Bowes melanoma although the protein moieties are substantiallysimilar. Differences in biological activities such as thermal stabilityand fibrin stimulatory properties were shown to be caused by thespecific glycoforms present.

The role of specific sugar units on the clearance of t-PA fromcirculation is further discussed, for example, by Lucore et al.,Circulation 77 (4), 906-914 (1988).

It is thus apparent that methods of modifying a glycoprotein'soligosaccharide structure can have substantial importance to proteinresearch for the development of biopharmaceuticals through carbohydrateengineering.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention a method for modifying aglycoprotein's oligosaccharide structure is provided which comprisessubstituting or deleting an amino acid residue at a position which isnon-adjacent or remote from the glycosylation site.

In a preferred embodiment of the invention, an amino acid substitutionis made at a cysteine residue to disrupt a disulfide bridge remote fromthe glycosylation site.

The method of the invention is especially adapted to modification of theoligosaccharide structure of plasminogen activators, namely urokinaseu-PA) and tissue plasminogen activator (t-PA). In particular, disruptionof a disulfide bridge in the growth factor domain can have a profoundeffect upon the oligosaccharide structure at a remote glycosylation sitein a kringle region. For example, disruption of a disulfide bridge inthe growth factor domain of t-PA can cause modification of theoligosaccharide structure at the glycosylation site in the kringle 1(KI) region, namely Asn-117, as well as at other glycosylation sitessuch as Asn-184 and Asn-448. Thus, in an illustrative example of thepresent invention, substituting an arginine residue for cysteine atamino acid position 73 in a recombinant t-PA produced in C-127 mousecells results in a modified oligosaccharide structure at Asn-117. Theoligosaccharide is changed from that of a high mannose type to a complextype oligosaccharide. This modified t-PA can be represented ast-PA[Cys(73)→Arg; Asn-117, complex oligosaccharide glycoform]or,alternatively, as F+GF[Cys(73)→Arg]+K1+K 2+SP. Similar such disruptionsof a disulfide bridge can be made by substituting an arginine residuefor cysteine at any of amino acid positions 51, 56, 62, 75 and 84 in thegrowth factor domain of t-PA, with substantially similar effects uponthe oligosaccharide structure at Asn-117. As used herein, the numberingof the 527 amino acid sequence of the normal t-PA molecule is thatdescribed by Pennica et al., Nature 301, 214-221 (1983).

In a like manner, disruption of a disulfide bridge in the growth factorregion of urokinase can produce modification of the oligosaccharidestructure at a remote glycosylation site of the molecule. For example,substituting an arginine residue for cysteine at amino acid position 42can have an effect on the oligosaccharide structure at Asn-302. As usedherein, the numbering of the 411 amino acid sequence of the normalurokinase molecule is that described by Holmes et al., Bio/Technology 3,923-929 (1985).

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following detailed description of preferredembodiments of the invention taken in conjunction with the appendeddrawings in which:

FIG. 1 is a graphical representation which shows the Bio-Gel P-4 columnchromatography profile of the oligosaccharides released byhydrazinolysis from (A) native t-PA (upper panel) and (B) modified t-PAF+GF[Cys(73)→Arg]+K1+K2+SP after neuraminidase digestion. The numbers atthe top indicate glucose units (g.u.). High mannose fractions areindicated with a bar whereas the complex oligosaccharide fractions aredesignated A, B, C and D. Radioactivity is shown on the vertical scaleand retention time in minutes on the horizontal scale.

FIG. 2 is a graphical representation which shows the Bio-Gel P-4 columnchromatography profile of the oligosaccharides isolated as in FIG. 1from a tryptic peptide containing the glycosylation site Asn-117 ofmodified t-PA F+GF[Cys(73)→Arg]+K1+K2+SP The complex oligosaccharidefractions are designated C and D and correspond to fractions C and D inpanel (B) of FIG. 1.

FIG. 3 (A) and (B) show the structures of the six major oligosaccharidesliberated from t-PA MB1023.

The invention is illustrated in one embodiment by the modified t-PAdisclosed in copending U.S. patent application Ser. No. 149,793, filedJan. 29, 1988, now U.S. Pat. No. 4,963,357, and assigned to a commonassignee. The modified t-PA was prepared from a chemically synthesizedgene coding for t-PA with a single point mutation of Arg for Cys atresidue 73. In this embodiment, also designated herein as t-PA variantMB1023, the mature protein has a 527 amino acid structure in whichresidue 73 is arginine instead of the cysteine that is present in nativet-PA. This variant was prepared by using an oligonucleotide sequence inthe construction of the synthetic gene which codes for Arg instead ofCys at the appropriate position.

The gene coding for this illustrative modified t-PA can be cloned intoand expressed in prokaryotic and eukaryotic hosts. For example, activemodified t-PA protein can be expressed in a prokaryotic host such as E.coli or a eukaryotic host such as Chinese hamster ovary (CHO) cells orC-127 mouse cells by operably inserting the modified t-PA codingsequence in replicable expression vectors or plasmids. For example, itcan be inserted into a suitable plasmid such as pML for production in E.coli and the bovine papilloma virus (BPV) vector for production in mousecells or a shuttle vector which can replicate in both prokaryotic andeukaryotic cells. In a preferred embodiment as used herein, the genecoding for the t-PA sequence t-PA[Cys(73)→Arg] was cloned into andexpressed from C-127 mouse cells. The excreted protein was extractedfrom the cell media by concentration and then purified on an affinitychromatography column.

A preferred cloning vector containing the nucleotide sequence for thet-PA variant MB1023 is plasmid pMON1401. This plasmid carried in a mouseC-127 host cell is on deposit with the American Type Culture Collection,Rockville, Md., under accession number ATCC CRL 9614. Other suitableeukaroytic and prokaryotic hosts for expression of the illustrativemodified t-PA used in this invention will be readily apparent to theperson skilled in the art after reading the present disclosure.

Determination of the structure of the oligosaccharides from theglycoprotein employs adaptation of the method used for ImmunoglobulinG-derived asparagine-linked oligosaccharides as described by Rademacherand Dwek, Prog. Immunol. 5, 95-112 (1983) and Parekh et al., Nature 316,452-457 (1985). According to this method, the glycoprotein sample issubjected to controlled hydrazinolysis to release intact its associatedoligosaccharide moieties as described by Takahasi et al., Meth. Enzymol.83, 263-268 (1982). Reduction of the reducing terminalN-acetylglucosamine residues using NaB³ H₄ is then performed to labelradioactively each carbohydrate chain. Each labeled oligosaccharidemixture is then subjected to exhaustive neuraminidase digestion in orderto analyze the distribution of neutral structures. The resulting`asialo` oligosaccharide mixtures are then fractionated by Bio-Gel® P-4(˜400 mesh) gel filtration chromatography, which separates neutraloligosaccharides on the basis of the effective hydrodynamic volumes asdescribed by Yamashita et al., Meth. Enzymol. 83 105-126 (1982). Bio-GelP-4 is a gel filtration material of choice for analysis of reducedoligosaccharides by gel permeation chromatography due to thepolyacrylamide structure. Bio-Gel P is prepared by copolymerization ofacrylamide with N,N'-methylene bis-acrylamide. P-4 has an exclusionlimit and fractionation range of about 800-4000 daltons. This well-knowngel filtration material is commercially available from Bio-RadLaboratories, Richmond, Calif.

The oligosaccharides also can be initially isolated from theglycoprotein by the method described in U.S. Pat. Nos. 4,719,294 and4,736,022. Said method employs hydrazinolysis of the glycoprotein underreaction conditions to cause cleavage at the N-linked sites, producing amixture having as a major component a de-N-acetylated hydrazonederivative of the oligosaccharides, followed by N-acylation of thehydrazone derivative, acid-catalysis of the hydrazone derivative toproduce unreduced oligosaccharides and subjecting the resultingunreduced oligosaccharides to cellulose column chromatography to removecontaminants and to recover the unreduced oligosaccharides. The lattermaterials, being essentially pure, can be used for attachment to variouspeptide or protein chains for further study.

Adaptation of these oligosaccharide structure determination methods tot-PA is disclosed in U.S. Pat. No. 4,751,084, the disclosure of which isincorporated herein by reference.

In an illustrative example of this invention, the modified t-PA MB1023and, as a control, native t-PA were subjected to hydrazinolysis,radiolabeled with sodium borotritide, treated with neuraminidase toremove sialic acid residues and co-chromatographed with unlabeleddextran polymers on Bio-Gel P-4. The P-4 patterns for theoligosaccharides of MB1023 are shown in FIG. 1. As can be seen, theMB1023 profile contains only a small amount of radioactiveoligosaccharide at glucose positions ranging from 9-12, corresponding tohigh mannose chains, whereas the native t-PA expressed in C-127 mousecells contains a substantial amount of high mannose oligosaccharides.

Determination of the structures of the six major oligosaccharidesliberated from t-PA MB1023 (shown in FIG. 3) was made by conventionalmethods of sequential exoglycosidase digestion as described in U.S. Pat.No. 4,751,084, Ex. 2. See also Kobata in "Biology of Carbohydrates,"Ginsburg and Robbins, Eds., John Wiley and Sons, pp. 87-162 (1984);Snider, Ibid., pp. 163-193. The following symbols are used to indicatemonosaccharide or other structural units and their residues in theoligosaccharides:

Galactose - Gal

Mannose - M

Fucose - F

N-Acetylglucosamine - Gn

The dash lines between monosaccharide units represent enzyme cleavagepoints. The six oligosaccharides shown belong to the bi- andtriantennary classes.

Tryptic peptides containing the glycosylation sites of the modified t-PAMB1023 were obtained by digestion of the glycoprotein with trypsin andfractionating the resulting mixture by reverse phase HPLC byconventional methods similar to those described, for example, by Pohl etal., Biochem. 23, 3701-3707 (1984); Vehar, Bio/Technology 2, 1051-1057(1984); and U.S. Pat. No. 4,751,084, Ex. 3. The tryptic glycopeptidefraction containing glycosylation site Asn-117 was subjected toprocedure for isolation of the oligosaccharide fractions, as above, andthe Bio-Gel P-4 pattern is shown in FIG. 2. As can be seen, the Asn-117site of MB1023 contains only a small amount of radioactiveoligosaccharide at those glucose positions corresponding to high mannosechains; whereas, it is known that the Asn-117 note in native t-PAcontains significant high mannose.

It will be appreciated that other mutations can be made to shift aglycosylation site of the glycoprotein from its normal position toanother potential glycosylation site and then a disulfide bridge can bedisrupted to modify the oligosaccharide structure as describedhereinbefore. For example, the glycosylation site in kringle 1 of t-PAcan be shifted from position 117 to 104 by mutating Asn-117→Ser andTrp-104→Asn, or shifted from position 117 to 125 by mutating Asn-117→Serand Pro-125→Asn, and then a disulfide bridge can be disrupted bymutating Cys-73→Arg.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed is:
 1. The method of modifying the oligosaccharidestructure of human t-PA which comprises substituting Arg for Cys at anyof amino acid residues 51, 56, 62, 73, 75 or 84 in said t-PA to therebymodify the oligosaccharide structure at Asn-117 from a high mannose typeto a complex type oligosaccharide structure and expressing said t-PA inC-127 cells.
 2. The method of claim 1 in which the substitution is madeat amino acid residue 73.